Method for the treatment of fibrosis

ABSTRACT

Disclosed is a method of treating fibrosis in a human or animal subject. The method comprises administering to the subject an effective amount of an antibody to an integrin or fragment thereof.

RELATED APPLICATIONS

This application is a continuation of prior U.S. provisional applicationSer. No. 60/130,847 filed on Apr. 22, 1999 as a continuation in part ofprior U.S. provisional application Ser. No. 60/137,214 filed on Jun. 1,1999.

FIELD OF THE INVENTION

This invention relates to methods for treating fibrosis in subjects inneed of such treatment.

BACKGROUND OF THE INVENTION

Collagen is a fibril-forming protein which is essential for maintainingthe integrity of the extracellular matrix found in connective tissues.The production of collagen is a highly regulated process, and itsdisturbance may lead to the development of tissue fibrosis. While theformation of fibrous tissue is part of the normal beneficial process ofhealing after injury, in some circumstances there is an abnormalaccumulation of fibrous materials such that it may ultimately lead toorgan failure (Border et al. (1994) New Engl. J. Med. 331:1286-1292).Injury to any organ leads to a stereotypical physiological response:platelet-induced hemostasis, followed by an influx of inflammatory cellsand activated fibroblasts. Cytokines derived from these cell types drivethe formation of new extracellular matrix and blood vessels (granulationtissue). The generation of granulation tissue is a carefullyorchestrated program in which the expression of protease inhibitors andextracellular matrix proteins is upregulated, and the expression ofproteases is reduced, leading to the accumulation of extracellularmatrix.

Central to the development of fibrotic conditions, whether induced orspontaneous, is stimulation of fibroblast activity. The influx ofinflammatory cells and activated fibroblasts into the injured organdepends on the ability of these cell types to interact with theinterstitial matrix comprised primarily of collagens. The major cellsurface collagen receptors are the α1β1 (VLA-1) and α2β1 (VLA-2)integrins. Both integrins have been implicated in cell adhesion andmigration on collagen (Keely et al. (1995) J. Cell Sci. 108:595-607 andGotwals et al. (1996) J. Clin. Invest. 97: 2469-2477); in promotingcontraction of collagen matrices (Gotwals et al. (1996) J. Clin. Invest.97: 2469-2477 and Schiro, (1991) Cell 67:403-410), and in regulating theexpression of genes involved in the remodeling of the extracellularmatrix (Riikonen et al. (1995) J. Biol. Chem. 270:1-5 and Langholz etal. (1995) J. Cell Biol. 131: 1903-1915). For example, when fibroblastscontact a collagen matrix, signaling through the α1β1 integrindown-regulates collagen I expression, while signaling through α2β1up-regulates the expression of matrix metalloproteases (Langholz et al.(1995) J. Cell Biol. 131: 1903-1915).

Many of the diseases associated with the proliferation of fibrous tissueare both chronic and often debilitating, including for example, skindiseases such as scleroderma. Some, including pulmonary fibrosis, can befatal due in part to the fact that the currently available treatmentsfor this disease have significant side effects and are generally notefficacious in slowing or halting the progression of fibrosis [Nagler etal. 1996, Am. J. Respir. Crit. Care Med., 154:1082-86].

There is, accordingly, a continuing need for new anti-fibrotic agents.

In contrast to the trends in research in the field of anti-fibrotictherapy which has focused on upstream cytokine mediators of fibrosis,such as TGF-B, we propose the use of antibody molecules comprisingantigen binding regions derived from the heavy or light chain variableregions of an anti-VLA antibody, for use in anti-fibrotic treatment andspecifically for treatment of pulmonary fibrosis.

SUMMARY OF THE INVENTION

The present invention provides a method of treating fibrosis in asubject. Specifically, the invention provides a method for treatingfibrosis, comprising administering to a patient a pharmaceuticalcomposition comprising an effective amount of an antibody moleculecomprising antigen binding regions derived from the light and heavychain variable regions of an anti-VLA antibody. In a preferredembodiment, the anti-VLA antibody is selected from the group consistingof anti-VLA-1, -2, -3, -4, -5, -6. In a most preferred embodiment, theinvention provides a method for treating pulmonary fibrosis, comprisingadministering to a patient a pharmaceutical composition, thepharmaceutical composition comprising an effective amount of an antibodymolecule comprising antigen binding regions derived from the light andheavy chain variable regions of an anti-VLA-1 and anti-VLA-2 antibody.

The anti-VLA antibody can be selected from the group consisting of ahuman antibody, a chimeric antibody, a humanized antibody and fragmentsthereof. The anti-VLA antibody can be a monoclonal or polyclonalantibody.

The invention further provides a method for treating fibrosis in asubject that is a human or animal subject.

All of the cited literature in the preceding section, as well as thecited literature included in the following disclosure, are herebyincorporated by reference.

DESCRIPTION OF THE DRAWINGS

FIG. 1. The α1-I domain binds collagen. A. Increasing concentrations ofthe human α1-I domain were bound to plates previously coated with 1μg/ml collagen I (squares) or collagen IV (circles). Values shown havebeen corrected for background binding to BSA. B. 2 μg/ml human α1-Idomain was mixed with increasing concentration of an anti-human α1β1integrin antibody 5E8D9 (squares) or an anti-human α2β1-integrinantibody A2IIE10 (circles), and then bound to plates previously coatedwith 1 μg/ml collagen IV. C. Plates were coated with 1 μg/ml collagen IVor 3% BSA. α1-I domain (2 μg/ml) was subsequenctly bound to coatedplates plates in the presence of 1 mM Mn²⁺, 1 mM Mg²⁺, or 5 mM EDTA.Data shown is representative of three independent experiments.

FIG. 2. Identification of a blocking mAb to the α1-I domain. A.Increasing concentration of mAbs AEF3 (triangles) or AJH10 (ATCC NO.______) (circles) were bound to plates coated with 30 μg/ml α1-I domain.B. The α1-I domain was treated with increasing concentrations of mAbAJH10 (ATCC NO. ______) (diamonds) or mAb BGC5 (squares) and boundcollagen IV (2 μg/ml) coated plates. C. K562-α1 cell were treated withincreasing concentration of mAbs AEF3(triangles) or AJH10 (ATCC NO.______) (circles) and bound to collagen IV (5 μg/ml) coated plates.45-50% of cells added to each well adhered to collagen IV. Data shown isrepresentative of three independent experiments.

FIG. 3. Species Cross-reactivity of the blocking mAbs. A. Detergentslysates from (1) sheep vascular smooth muscle, (2) human leukemiaK562-α1 cells or (3) purified RΔH GST-I domain; (4) Rat GST-α1 I domain;and (5) human GST-α1 I domain were separated by 10-20% SDS-PAGE undernon-reducing conditions, and immunoblotted with function-blocking mAbAJH10 (ATCC NO. ______). Molecular weight markers are shown on the left;non-reduced α1β1 integrin migrates at ˜180 kDa; GST-I domain migrates at˜45 kDa. B. Rabbit vascular smooth muscle cells were incubated witheither mAb AJH10 (ATCC NO. ______)(bottom) or murine IgG control (top)and analyzed by fluorescence activated cell sorter (FACS).

FIG. 4. Location of the Epitope for the anti-α1 I domain Blocking mAbs.A. Amino acid sequence of the rat (top) and human (below) α1-I domain.The residues that comprise the MIDAS (metal ion dependent adhesion site)motif are shown in bold. The human amino acids that replaced thecorresponding rat residues (RΔH) are shown below the rat sequence in theboxed region. For clarity, residue numbering in the text refers to thisfigure. B. Increasing concentrations of mAb AJH10 (ATCC NO. ______) werebound to plates coated with 30 μg/ml human (circles), rat (triangles) orRΔH (squares) α1-I domain. Data shown is representative of threeexperiments.

FIG. 5. Amino acid sequence of the human α1-I domain.

FIG. 6. Cation Stabilizes the Expression of the Epitope. A. 0.5 μg ofblocking mAb AJH10 (ATCC NO. ______) or non-blocking mAb AEF3 in thepresence of 5 mM EDTA (open) or 1 mM MnCl₂ (solid) were bound to platespreviously coated with 1 μg/ml affinity purified, human α1β1 integrin.B. 5 μg/ml AJH10 (ATCC NO. ______) or AEF3 were incubated with K562-α1cells in the presence of 2 mM MnCl₂ (solid), or following a wash with 5mM EDTA (open). Bound antibody was measured by FACS and is reported asthe mean fluorescence intensity (MFI).

FIG. 7. Denaturation of the α1-I domain by Urea. 0.6 μM rat α1 I domain,in the presence of no cation (squares), or 1 mM MnCl₂ (circles) andincreasing concentrations of urea were analyzed at 25° C. using anexcitation wavelength of 280 nm. Fluorescence data from the emissionspectra at 350 nm are plotted as a function of urea concentration andstandardized using the change in fluorescence for each of the testconditions as a measure of the total fraction unfolded.

FIG. 8. Circular dichroism spectra of thermally denatured α1-I domain.Temperature dependent, circular dichroism measurements at fixedwavelength (222 nM) were performed using 55 μM α1-I domain in theabsence (solid line), or presence of 2 mM Mg²⁺ (dot-dash line), or 2 mMMn²⁺ (dotted line). Data are expressed as (A) continuous temperaturedependence of molar ellipticity per residue, and (B) first derivativecurves after smoothing the corresponding data curves shown in panel A.

DETAILED DESCRIPTION OF THE INVENTION

The present application is directed to the discovery that antibodies tointegrins and fragments thereof can be used for the treatment offibrosis.

The methods of the present invention contemplate the use of antibodiesto integrins where the integrins contemplated include molecules whichcomprise a β chain, including but not limited to β1, β2, β3, β4, β5, β6,β7, β8, non-covalently bound to an α chain, including but not limited toα1, α2, α3, α4, α5, α6, α7, α8, α9, α10, αV, αL, αM, αX, αD, αE, αIIb.Examples of the various integrins contemplated for use in the inventioninclude, but are not limited to:

-   -   α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, α9β1, α10β1,        αVβ1, αLβ1, αMβ1, αXβ1, αDβ1, αIIbβ1, αEβ1;    -   α1β2, α2β2, α3β2, α4β2, α5β2, α6β2, α7β2, α8β2, α9β2, α10β2,        αVβ2, αLβ2, αMβ2, αXβ2, αDβ2, αIIbβ2, αEβ2;    -   α1β3, α2β3, α3β3, α4β3, α5β3, α6β3, α7β3, α8β3, α9β3, α10β3,        αVβ3, αLβ3, αMβ3, αX,β3, αDβ3, αIIbβ3, αEβ3;    -   α1β4, α2β4, α3β4, α4β4, α5β4, α6β4, α7β4, α8β4, α9β4, α10β4,        αVβ4, αLβ4, αMβ4, αXβ4 αDβ4, αIIbβ4, αEβ4;    -   α1β5, α2β5, α3β5, α4β5, α5β5, α6β5, α7β5, α8β5, α9β5, α10β5,        αVβ5, αLβ5, αMβ5, αXβ5, αDβ5, αIIbβ5, αEβ5;    -   α1β6, α2β6, α3β6, α4β6, α5β6, α6β6, α7β6, α8β6, α9β6, α10β6,        αVβ6, αLβ6, αMβ6, αXβ6, αDβ6, αIIbβ6, αEβ6;    -   α1β7, α2β7, α3β7, α4β7, α5β7, α6β7, α7β7, α8β7, α9β7, α10β7,        αVβ7, αLβ7, αMβ7, αXβ7, αDβ7, αIIbβ7, αEβ7;    -   α1β8, α2β8, α3β8, α4β8, α5β8, α6β8, α7β8, α8β8, α9β8, α10β8,        αVβ8, αLβ8, αMβ8, αXβ8, αDβ8, αIIbβ8, αEβ8;

The methods of the present invention also contemplate the use ofantibodies to integrin fragments including for example antibodies to a βchain alone, including but not limited to β1, β2, β3, β4, β5, β6, β7,β8, as well as an a chain alone, including but not limited to α1, α2,α3, α2, α5, α6, α7, α7, α8, α9, α10, αV, αL, αM, αX, αD, αE, αIIb. Inaddition, the methods of the present invention further contemplate theuse of antibodies to integrin fragments including for example antibodiesto the I domain of the α chain, including but not limited to the Idomain from α1β1 (Briesewitz et al., 1993 J. Biol. Chem. 268:2989); α2β1(Takada and Hemler, 1989 J Cell Biol 109:397), αLβ2 (Larson et al., 1989J Cell Biol 108:703), αMβ2 (Corbi et al., 1988 J Biol Chem 263:12403),αXβ2 (Corbi et al., 1987 EMBO J 6:4023), αDβ2 (Grayson et al., 1988 JExp Med 188:2187), αEβ7 (Shaw et al., 1994 J Biol Chem 269:6016). In apreferred embodiment, the alpha1-I domain antigenic determinantcomprises an amino acid sequence of at least 6 contiguous amino acids,wherein the contiguous sequence is found within the sequence of FIG. 5.Moreover, in a preferred embodiment, the contiguous sequence isVal-Gln-Arg-Gly-Gly-Arg.

In a preferred embodiment the invention contemplates the use ofantibodies to VLA-1, -2, -3, -4, -5, -6, in which each of the moleculescomprise a β1 chain non covalently bound to a α chain, (α1, α2, α3, α4,α5, α6), respectively. In a most preferred embodiment, the inventioncontemplates using anti-VLA-1 and anti-VLA-2 antibodies for thetreatment of pulmonary fibrosis.

Methods for producing integrins for use in the present invention areknown to those of skill in the art (see for e.g. Springer et al. 1990,Nature 346:425-434).

Embodiments of the present invention include polyclonal and monoclonalantibodies to integrins and fragments thereof. Preferred embodiments ofthe present invention include a monoclonal antibody, including forexample, an anti-VLA antibody homolog. Preferred antibodies and homologsfor treatment, in particular for human treatment, include human antibodyhomologs, humanized antibody homologs, chimeric antibody homologs, Fab,Fab′, F(ab′)2 and F(v) antibody fragments, and monomers or dimers ofantibody heavy or light chains or mixtures thereof. Thus, monoclonalantibodies against an integrin molecule or fragment thereof are thepreferred binding agent in the method of the invention.

As used herein, the term “antibody homolog” includes intact antibodiesconsisting of immunoglobulin light and heavy chains linked via disulfidebonds. The term “antibody homolog” is also intended to encompass aprotein comprising one or more polypeptides selected from immunoglobulinlight chains, immunoglobulin heavy chains and antigen-binding fragmentsthereof which are capable of binding to one or more antigens (i.e.,VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6). The component polypeptides ofan antibody homolog composed of more than one polypeptide may optionallybe disulfide-bound or otherwise covalently crosslinked.

Accordingly, therefore, “antibody homologs” include intactimmunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof), wherein the light chains of the immunoglobulin may be of typeskappa or lambda. “Antibody homologs” also include portions of intactantibodies that retain antigen-binding specificity, for example, Fabfragments, Fab′ fragments, F(ab′)2 fragments, F(v) fragments, heavychain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like. Thus,antigen-binding fragments, as well as full-length dimeric or trimericpolypeptides derived from the above-described antibodies are themselvesuseful.

As used herein, a “humanized antibody homolog” is an antibody homolog,produced by recombinant DNA technology, in which some or all of theamino acids of a human immunoglobulin light or heavy chain that are notrequired for antigen binding have been substituted for the correspondingamino acids from a nonhuman mammalian immunoglobulin light or heavychain.

As used herein, a “chimeric antibody homolog” is an antibody homolog,produced by recombinant DNA technology, in which all or part of thehinge and constant regions of an immunoglobulin light chain, heavychain, or both, have been substituted for the corresponding regions fromanother immunoglobulin light chain or heavy chain. In another aspect theinvention features a variant of a chimeric molecule which includes: (1)a VLA targeting moiety; (2) optionally, a second peptide, e.g., onewhich increases solubility or in vivo life time of the VLA targetingmoiety, e.g., a member of the immunoglobulin super family or fragment orportion thereof, e.g., a portion or a fragment of IgG, e.g., the humanIgGl heavy chain constant region, e.g., CH2 and CH3 hinge regions; and atoxin moiety. The chimeric molecule can be used to treat a subject,e.g., a human, at risk for disorder related to proliferation ofepithelial cells such as hair follicles and the like.

As used herein, a “human antibody homolog” is an antibody homologproduced by recombinant DNA technology, in which all of the amino acidsof an immunoglobulin light or heavy chain that are derived from a humansource.

A “a subject with a fibrotic condition” refers to, but is not limitedto, subjects afflicted with fibrosis of an internal organ, subjectsafflicted with a dermal fibrosing disorder, and subjects afflicted withfibrotic conditions of the eye.

Fibrosis of internal organs (e.g., liver, lung, kidney, heart bloodvessels, gastrointestinal tract), occurs in disorders such as pulmonaryfibrosis, myelofibrosis, liver cirrhosis, mesangial proliferativeglomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy,renal interstitial fibrosis, renal fibrosis in patients receivingcyclosporin, and HIV associated nephropathy. In a preferred embodiment,the invention contemplates using the anti-VLA antibodies for thetreatment of pulmonary fibrosis. In a most preferred embodiment, theinvention contemplates using anti-VLA-1 and anti-VLA-2 antibodies forthe treatment of pulmonary fibrosis.

Dermal fibrosing disorders include, but are not limited to, scleroderma,morphea, keloids, hypertrophic scars, familial cutaneous collagenoma,and connective tissue nevi of the collagen type.

Fibrotic conditions of the eye include conditions such as diabeticretinopathy, postsurgical scarring (for example, after glaucomafiltering surgery and after cross-eye surgery), and proliferativevitreoretinopathy.

Additional fibrotic conditions which may be treated by the methods ofthe present invention include: rheumatoid arthritis, diseases associatedwith prolonged joint pain and deteriorated joints; progressive systemicsclerosis, polymyositis, dermatomyositis, eosinophilic fascitis,morphea, Raynaud's syndrome, and nasal polyposis.

In addition, fibrotic conditions which may be treated the methods ofpresent invention also include inhibiting overproduction of scarring inpatients who are known to form keloids or hypertrophic scars, inhibitingor preventing scarring or overproduction of scarring during healing ofvarious types of wounds including surgical incisions, surgical abdominalwounds and traumatic lacerations, preventing or inhibiting scarring andreclosing of arteries following coronary angioplasty, preventing orinhibiting excess scar or fibrous tissue formation associated withcardiac fibrosis after infarction and in hypersensitive vasculopathy.

An “effective amount” (when used in the toleragenic context) is anamount sufficient to effect beneficial or desired clinical results. Aneffective amount can be administered in one or more administrations. Interms of treatment, an “effective amount” of an antibody for use in thepresent invention, including for example an anti-VLA antibody, is anamount sufficient to palliate, ameliorate, stabilize, reverse, slow ordelay progression of a fibrotic condition in accordance with clinicallyacceptable standards for disorders to be treated or for cosmeticpurposes. Detection and measurement of indicators of efficacy may bemeasured by a number of available diagnostic tools, including, forexample, by physical examination including blood tests, pulmonaryfunction tests, and chest X-rays; CT scan; bronchoscopy; bronchoalveolarlavage; lung biopsy and CT scan.

Methods of Making Antibodies

The technology for producing monoclonal antibodies, including forexample, anti-integrin monoclonal antibodies is well known. See forexample, Mendrick et al. 1995, Lab. Invest. 72:367-375 (mAbs to murineanti-α1β1 and anti-α2β1); Sonnenberg et al. 1987 J. Biol. Chem.262:10376-10383 (mAbs to murine anti-α6β1); Yao et al. 1996, J Cell Sci1996 109:3139-50 (mAbs to murine anti-α7β1); Hemler et al. 1984, JImmunol 132:3011-8 (mAbs to human α1β1); Pischel et al. 1987 J Immunol138:226-33 (mAbs to human α2β1); Wayner et al. 1988, J Cell Biol107:1881-91 (mAbs to human α3β1); Hemler et al. 1987 J Biol Chem262:11478-85 (mAbs to human α4β1); Wayner et al. 1988 J. Cell Biol107:1881-91 (mAbs to human α5β1); Sonnenberg et al. 1987, J. Biol. Chem.262:10376-10383 (mAbs to human α6β1); A Wang et al. 1996 Am. J. Respir.Cell Mol Biol. 15:664-672 (mAbs to human α9β1); Davies et al. 1989 JCell Biol 109:1817-26 (mAbs to human αVβ1); Sanchez-Madrid et al. 1982,Proc Natl Acad Sci USA 79:7489-93 (mAbs to human αLβ2); Diamond et al.1993, J Cell Biol 120:1031-43 (mAbs to human αMβ2); Stacker et al. 1991J Immunol 146:648-55 (mAbs to human αXβ2); Van der Vieren et al 1995Immunity 3:683-90 (mAbs to human αDβ2); Bennett et al. 1983 Proc NatlAcad Sci USA 80:2417-21 (mAbs to human αIIbβ2); Hessle et al. 1984,Differentiation 26:49-54 (mAbs to human α6β4); Weinacker et al. 1994 JBiol Chem 269:6940-8 (mAbs to human αVβ5); Weinacker et al. 1994 J BiolChem 269:6940-8 (mAbs to human αVβ6); Cerf-Bensussan et al 1992 Eur JImmunol 22:273-7 (mAbs to human αEβ7); Nishimura et al. 1994 J Biol Chem269:28708-15 (mAbs to human αVβ8); Bossy et al. 1991 EMBO J 10:2375-85(polyclonal antisera to human α8β1); Camper et al. 1998 J. Biol. Chem.273:20383-20389 (polyclonal antisera to human α10β1).

In general, an immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. Preferred immortalcell lines are mouse myeloma cell lines that are sensitive to culturemedium containing hypoxanthine, arninopterin and thymidine (“HATmedium”). Typically, HAT-sensitive mouse myeloma cells are fused tomouse splenocytes using 1500 molecular weight polyethylene glycol (“PEG1500”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively ftised myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridomas producing a desired antibody are detected byscreening the hybridoma culture supernatants. For example, hybridomasprepared to produce anti-VLA antibodies may be screened by testing thehybridoma culture supernatant for secreted antibodies having the abilityto bind to a recombinant VLA-expressing cell line.

To produce anti-VLA antibody homologs that are intact immunoglobulins,hybridoma cells that tested positive in such screening assays werecultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. Tissue culture techniques and culture media suitablefor hybridoma cells are well known. The conditioned hybridoma culturesupernatant may be collected and the anti-VLA antibodies optionallyfurther purified by well-known methods.

Alternatively, the desired antibody may be produced by injecting thehybridoma cells into the peritoneal cavity of an unimmunized mouse. Thehybridoma cells proliferate in the peritoneal cavity, secreting theantibody which accumulates as ascites fluid. The antibody may beharvested by withdrawing the ascites fluid from the peritoneal cavitywith a syringe.

Fully human monoclonal antibody homologs against VLA are anotherpreferred binding agent which may block antigens in the method of theinvention. In their intact form these may be prepared using invitro-primed human splenocytes, as described by Boerner et al., 1991, J.Inmmunol. 147:86-95, “Production of Antigen-specific Human MonoclonalAntibodies from In Vitro-Primed Human Splenocytes”.

Alternatively, they may be prepared by repertoire cloning as describedby Persson et al., 1991, Proc. Nat. Acad. Sci. USA 88: 2432-2436,“Generation of diverse high-affinity human monoclonal antibodies byrepertoire cloning” and Huang and Stollar, 1991, J. Immunol. Methods141: 227-236, “Construction of representative immunoglobulin variableregion CDNA libraries from human peripheral blood lymphocytes without invitro stimulation”. U.S. Pat. No. 5,798,230 (Aug. 25, 1998, “Process forthe preparation of human monoclonal antibodies and their use”) describespreparation of human monoclonal antibodies from human B cells. Accordingto this process, human antibody-producing B cells are immortalized byinfection with an Epstein-Barr virus, or a derivative thereof, thatexpresses Epstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2 function,which is required for immortalization, is subsequently shut off, whichresults in an increase in antibody production.

In yet another method for producing fully human antibodies, U.S. Pat.No. 5,789,650 (Aug. 4, 1998, “Transgenic non-human animals for producingheterologous antibodies”) describes transgenic non-human animals capableof producing heterologous antibodies and transgenic non-human animalshaving inactivated endogenous immunoglobulin genes. Endogenousimmunoglobulin genes are suppressed by antisense polynucleotides and/orby antiserum directed against endogenous immunoglobulins. Heterologousantibodies are encoded by immunoglobulin genes not normally found in thegenome of that species of non-human animal. One or more transgenescontaining sequences of unrearranged heterologous human immunoglobulinheavy chains are introduced into a non-human animal thereby forming atransgenic animal capable of functionally rearranging transgenicimmunoglobulin sequences and producing a repertoire of antibodies ofvarious isotypes encoded by human immunoglobulin genes. Suchheterologous human antibodies are produced in B-cells which arethereafter immortalized, e.g., by fusing with an immortalizing cell linesuch as a myeloma or by manipulating such B-cells by other techniques toperpetuate a cell line capable of producing a monoclonal heterologous,fully human antibody homolog.

Yet another preferred binding agent which may block VLA antigens in themethod of the invention is a humanized antibody homolog having thecapability of binding to a VLA protein. Following the early methods forthe preparation of chimeric antibodies, a new approach was described inEP 0239400 (Winter et al.) whereby antibodies are altered bysubstitution of their complementarity determining regions (CDRs) for onespecies with those from another. This process may be used, for example,to substitute the CDRs from human heavy and light chain Ig variableregion domains with alternative CDRs from murine variable regiondomains. These altered Ig variable regions may subsequently be combinedwith human Ig constant regions to created antibodies which are totallyhuman in composition except for the substituted murine CDRs. SuchCDR-substituted antibodies would be predicted to be less likely toelicit an immune response in humans compared to chimeric antibodiesbecause the CDR-substituted antibodies contain considerably lessnon-human components. The process for humanizing monoclonal antibodiesvia CDR “grafting” has been termed “reshaping”. (Riechmann et al., 1988Nature 332: 323-327, “Reshaping human antibodies for therapy”; Verhoeyenet al., 1988, Science 239: 1534-1536, “Reshaping of human antibodiesusing CDR-grafting in Monoclonal Antibodies”.

Typically, complementarity determining regions (CDRs) of a murineantibody are transplanted onto the corresponding regions in a humanantibody, since it is the CDRs (three in antibody heavy chains, three inlight chains) that are the regions of the mouse antibody which bind to aspecific antigen. Transplantation of CDRs is achieved by geneticengineering whereby CDR DNA sequences are determined by cloning ofmurine heavy and light chain variable (V) region gene segments, and arethen transferred to corresponding human V regions by site directedmutagenesis. In the final stage of the process, human constant regiongene segments of the desired isotype (usually gamma I for CH and kappafor CL) are added and the humanized heavy and light chain genes areco-expressed in mammalian cells to produce soluble humanized antibody.

The transfer of these CDRs to a human antibody confers on this antibodythe antigen binding properties of the original murine antibody. The sixCDRs in the murine antibody are mounted structurally on a V region“framework” region. The reason that CDR-grafting is successful is thatframework regions between mouse and human antibodies may have verysimilar 3-D structures with similar points of attachment for CDRS, suchthat CDRs can be interchanged. Such humanized antibody homologs may beprepared, as exemplified in Jones et al., 1986 Nature 321: 522-525,“Replacing the complementarity-determining regions in a human antibodywith those from a mouse”; Riechmann, 1988, Nature 332:323-327,“Reshaping human antibodies for therapy”; Queen et al., 1989, Proc. Nat.Acad. Sci. USA 86:10029, “A humanized antibody that binds to theinterleukin 2 receptor” and Orlandi et al., 1989, Proc. Natl. Acad. Sci.USA 86:3833 “Cloning Immunoglobulin variable domains for expression bythe polymerase chain reaction”.

Nonetheless, certain amino acids within framework regions are thought tointeract with CDRs and to influence overall antigen binding affinity.The direct transfer of CDRs from a murine antibody to produce ahumanized antibody without any modifications of the human V regionframeworks often results in a partial or complete loss of bindingaffinity. In a number of cases, it appears to be critical to alterresidues in the framework regions of the acceptor antibody in order toobtain binding activity.

Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86: 10029-10033, “Ahumanized antibody that binds to the interleukin 2 receptor” and WO90/07861 (Protein Design Labs Inc.) have described the preparation of ahumanized antibody that contains modified residues in the frameworkregions of the acceptor antibody by combining the CDRs of a murine mAb(anti-Tac) with human immunoglobulin framework and constant regions.They have demonstrated one solution to the problem of the loss ofbinding affinity that often results from direct CDR transfer without anymodifications of the human V region framework residues; their solutioninvolves two key steps. First, the human V framework regions are chosenby computer analysts for optimal protein sequence homology to the Vregion framework of the original murine antibody, in this case, theanti-Tac MAb. In the second step, the tertiary structure of the murine Vregion is modelled by computer in order to visualize framework aminoacid residues which are likely to interact with the murine CDRs andthese murine amino acid residues are then superimposed on the homologoushuman framework. Their approach of employing homologous human frameworkswith putative murine contact residues resulted in humanized antibodieswith similar binding affinities to the original murine antibody withrespect to antibodies specific for the interleukin 2 receptor (Queen etal., 1989 [supra]) and also for antibodies specific for herpes simplexvirus (HSV) (Co. et al., 1991, Proc. Nat. Acad. Sci. USA 88: 2869-2873,“Humanised antibodies for antiviral therapy”.

According to the above described two step approach in WO 90/07861, Queenet al. outlined several criteria for designing humanizedimmunoglobulins. The first criterion is to use as the human acceptor theframework from a particular human immunoglobulin that is usuallyhomologous to the non-human donor immunoglobulin to be humanized, or touse a consensus framework from many human antibodies. The secondcriterion is to use the donor amino acid rather than the acceptor if thehuman acceptor residue is unusual and the donor residue is typical forhuman sequences at a specific residue of the framework. The thirdcriterion is to use the donor framework amino acid residue rather thanthe acceptor at positions immediately adjacent to the CDRS

One may use a different approach (see Tempest, 1991, Biotechnology 9:266-271, “Reshaping a human monoclonal antibody to inhibit humanrespiratory syncytial virus infection in vivo”) and utilize, asstandard, the V region frameworks derived from NEWM and REI heavy andlight chains respectively for CDR-grafting without radical introductionof mouse residues. An advantage of using the Tempest et al., 1991approach to construct NEWM and REI based humanized antibodies is thatthe 3dimensional structures of NEWM and REI variable regions are knownfrom x-ray crystallography and thus specific interactions between CDRsand V region framework residues can be modeled.

Regardless of the approach taken, the examples of the initial humanizedantibody homologs prepared to date have shown that it is not astraightforward process. However, even acknowledging that such frameworkchanges may be necessary, it is not possible to predict, on the basis ofthe available prior art, which, if any, framework residues will need tobe altered to obtain functional humanized antibodies of the desiredspecificity. Results thus far indicate that changes necessary topreserve specificity and/or affinity are for the most part unique to agiven antibody and cannot be predicted based on the humanization of adifferent antibody.

Subjects

The subject treatments are effective on both human and animal subjectsafflicted with these conditions. Animal subjects to which the inventionis applicable extend to both domestic animals and livestock, raisedeither as pets or for commercial purposes. Examples are dogs, cats,cattle, horses, sheep, hogs and goats.

Pharmaceutical Preparations

In the methods of the invention the anti-VLA antibodies may beadministered parenterally. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

The antibody homologs are preferably administered as a sterilepharmaceutical composition containing a pharmaceutically acceptablecarrier, which may be any of the numerous well known carriers, such aswater, saline, phosphate buffered saline, dextrose, glycerol, ethanol,and the like, or combinations thereof. The compounds of the presentinvention may be used in the form of pharmaceutically acceptable saltsderived from inorganic or organic acids and bases. Included among suchacid salts are the following: acetate, adipate, alginate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include anmmonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine and saltswith amino acids such as arginine, lysine, and so forth. Also, the basicnitrogen-containing groups can be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride,bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl,dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides, aralkyl halides,such as benzyl and phenethyl bromides and others. Water or oil-solubleor dispersible products are thereby obtained.

The pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, or pharmaceutically acceptablederivatives thereof, together with any pharmaceutically acceptablecarrier. The term “carrier” as used herein includes acceptable adjuvantsand vehicles. Pharmaceutically acceptable carriers that may be used inthe pharmaceutical compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

According to this invention, the pharmaceutical compositions may be inthe form of a sterile injectable preparation, for example a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as do naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions.

The pharmaceutical compositions of this invention may be given orally.If given orally, they can be administered in any orally acceptabledosage form including, but not limited to, capsules, tablets, aqueoussuspensions or solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions are required for oral use,the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening, flavoring or coloring agents mayalso be added.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. Topically-transdermalpatches may also be used. For topical applications, the pharmaceuticalcompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, the pharmaceuticalcompositions can be formulated in a suitable lotion or cream containingthe active components suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

In addition to the direct topical application of the preparations theycan be topically administered by other methods, for example,encapsulated in a temperature and/or pressure sensitive matrix or infilm or solid carrier which is soluble in body fluids and the like forsubsequent release, preferably sustained-release of the activecomponent. As appropriate compositions for topical application there maybe cited all compositions usually employed for topically administeringtherapeutics, e.g., creams, genies, dressings, shampoos, tinctures,pastes, ointments, salves, powders, liquid or semiliquid formulation andthe like. Application of said compositions may be by aerosol e.g. with apropellent such as nitrogen carbon dioxide, a freon, or without apropellent such as a pump spray, drops, lotions, or a semisolid such asa thickened composition which can be applied by a swab. In particularcompositions, semisolid compositions such as salves, creams, pastes,genies, ointments and the like will conveniently be used.

Particular compositions for use in the method of the present inventionare those wherein the anti-VLA antibody is formulated in vesicles suchas liposome-containing compositions. Liposomes are vesicles formed byamphiphatic molecules such as polar lipids, for example, phosphatidylcholines, ethanolamines and serines, sphingomyelins, cardiolipins,plasmalogens, phosphatidic acids and cerebiosides. Liposomes are formedwhen suitable amphiphathic molecules are allowed to swell in water oraqueous solutions to form liquid crystals usually of multilayerstructure comprised of many bilayers separated from each other byaqueous material (also referred to as coarse liposomes). Another type ofliposome known to be consisting of a single bilayer encapsulatingaqueous material is referred to as a unilamellar vesicle. Ifwatersoluble materials are included in the aqueous phase during theswelling of the lipids they become entrapped in the aqueous layerbetween the lipid bilayers.

A particularly convenient method for preparing liposome formulated formsof anti-VLA antibodies is the method described in EP-A-253,619,incorporated herein by reference. In this method, single bilayeredliposomes containing encapsulated active ingredients are prepared bydissolving the lipid component in an organic medium, injecting theorganic solution of the lipid component under pressure into an aqueouscomponent while simultaneously mixing the organic and aqueous componentswith a high speed homogenizer or mixing means, whereupon the liposomesare formed spontaneously. The single bilayered liposomes containing theencapsulated active ingredient can be employed directly or they can beemployed in a suitable pharmaceutically acceptable carrier for topicaladministration. The viscosity of the liposomes can be increased by theaddition of one or more suitable thickening agents such as, for examplexanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose andmixtures thereof. The aqueous component may consist of water alone or itmay contain electrolytes, buffered systems and other ingredients, suchas, for example, preservatives. Suitable electrolytes which can beemployed include metal salts such as alkali metal and alkaline earthmetal salts. The preferred metal salts are calcium chloride, sodiumchloride and potassium chloride. The concentration of the electrolytemay vary from zero to 260 mM, preferably from 5 mM to 160 mM. Theaqueous component is placed in a suitable vessel which can be adapted toeffect homogenization by effecting great turbulence during the injectionof the organic component. Homogenization of the two components can beaccomplished within the vessel, or, alternatively, the aqueous andorganic components may be injected separately into a mixing means whichis located outside the vessel. In the latter case, the liposomes areformed in the mixing means and then transferred to another vessel forcollection purpose.

The organic component consists of a suitable non-toxic, pharmaceuticallyacceptable solvent such as, for example ethanol, glycerol, propyleneglycol and polyethylene glycol, and a suitable phospholipid which issoluble in the solvent. Suitable phospholipids which can be employedinclude lecithin, phosphatidylcholine, phosphatydylserine,phosphatidylethanol-amine, phosphatidylinositol, lysophosphatidylcholineand phospha-tidyl glycerol, for example. Other lipophilic additives maybe employed in order to selectively modify the characteristics of theliposomes. Examples of such other additives include stearylamine,phosphatidic acid, tocopherol, cholesterol and lanolin extracts.

In addition, other ingredients which can prevent oxidation of thephospholipids may be added to the organic component. Examples of suchother ingredients include tocopherol, butylated hydroxyanisole,butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.Preservatives such a benzoic acid, methyl paraben and propyl paraben mayalso be added.

Apart from the above-described compositions, use may be made of covers,e.g. plasters, bandages, dressings, gauze pads and the like, containingan appropriate amount of an anti-VLA antibody therapeutic. In some casesuse may be made of plasters, bandages, dressings, gauze pads and thelike which have been impregnated with a topical formulation containingthe therapeutic formulation.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation through the use of anebulizer, a dry powder inhaler or a metered dose inhaler. Suchcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and may be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, fluorocarbons, and/orother conventional solubilizing or dispersing agents.

According to another embodiment compositions containing a compound ofthis invention may also comprise an additional agent selected from thegroup consisting of corticosteroids, antiinflammatories,immunosuppressants, antimetabolites, and immunomodulators. Specificcompounds within each of these classes may be selected from any of thoselisted under the appropriate group headings in “Comprehensive MedicinalChemistry”, Pergamon Press, Oxford, England, pp. 970-986 (1990), thedisclosure of which is herein incorporated by reference. Also includedwithin this group are compounds such as theophylline, sulfasalazine andaminosalicylates (antiinflammatories); cyclosporin, FK-506, andrapamycin (immunosuppressants); cyclophosphamide and methotrexate(antimetabolites); steroids (inhaled, oral or topical) and interferons(immunomodulators).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated, and the particular mode of administration. It should beunderstood, however, that a specific dosage and treatment regimen forany particular patient will depend upon a variety of factors, includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, rate of excretion,drug combination, and the judgment of the treating physician and theseverity of the particular disease being treated. The amount of activeingredient may also depend upon the therapeutic or prophylactic agent,if any, with which the ingredient is co-administered.

The dosage and dose rate of the compounds of this invention effective toproduce the desired effects will depend on a variety of factors, such asthe nature of the inhibitor, the size of the patient, the goal of thetreatment, the nature of the pathology to be treated, the specificpharmaceutical composition used, and the judgment of the treatingphysician. Dosage levels of between about 0.001 and about 100 mg/kg bodyweight per day, preferably between about 0.1 and about 50 mg/kg bodyweight per day of the active ingredient compound are useful. Mostpreferably, the antibody homlogs will be administered at a dose rangingbetween about 0.1 mg/kg body weight/day and about 20 mg/kg bodyweight/day, preferably ranging between about 0.1 mg/kg body weight/dayand about 10 mg/kg body weight/day and at intervals of every 1-14 days.Preferably, an antibody composition is administered in an amounteffective to provide a plasma level of antibody of at least 1 μg/ml.

Persons having ordinary skill in the art can readily test if anantagonist of the invention is having it intended effect. For instance,cells contained in a sample of the individual's epithelium are probedfor the presence of the agent in vitro (or ex vivo) using a secondreagent to detect the administered agent. For example, this may be afluorochrome labelled antibody specific for the administered agent whichis then measured by standard FACS (fluorescence activated cell sorter)analysis. Alternatively, presence of the administered agent is detectedin vitro (or ex vivo) by the inability or decreased ability of theindividual's cells to bind the same agent which has been itself labelled(e.g., by a fluorochrome). The preferred dosage should producedetectable coating of the vast majority of hedgehog-positive cells.Preferably, coating is sustained in the case of an antibody homolog fora 1-14 day period.

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning: ALaboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.),Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II(D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.),1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcriptionand Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture ofAnimal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; ImmobilizedCells and Enzymes, IRL Press, 1986; A Practical Guide to MolecularCloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155(Wu et al., eds), Academic Press, New York; Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold SpringHarbor Laboratory; Immunoclemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), Academic Press, London, 1987; Handbook ofExperiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds.), 1986; Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, 1986.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLES Example 1

Treatment of Animals

Male C57/BL6 mice weighing 28-30 g, were housed in plastic cages ingroups of 4 in facilities approved by the American Association forAccreditation of Laboratory Animal Care. The animals were allowed toacclimate for one week to laboratory conditions prior to starting theexperiments. They had access to Rodent Laboratory Chow 5001 (PurinaMills, Inc., St. Louis, Mo.) and water ad libitum and housed in a roomwhich gets filtered air and has 12 hr light /12 hr dark cycle. Mice wereassigned into the following groups: GROUP TREATMENT A Saline + Phosphatebuffered saline B Saline + Ha4/8 control IgG C Bleomycin + Ha4/8 controlIgG D Bleomycin + Ha31/8 (hamster anti-α1 β1 rat integrin antibody) EBleomycin + Ha1/29 (hamster anti-α2 β1 rat integrin antibody)

Bleomycin sulfate was dissolved in pyrogen free sterile isotonic salinejust before intratraceheal (IT) instillation. Under methoxyfluraneanaesthesia mice in appropriate groups received by intratrachealadministration either 100 μl of sterile isotonic solution or 0.08 unitsof bleomycin solution in 100 μl. Antibodies (4 mg/kg) were administeredby intraperitoneal injection to mice in appropriate groups three times aweek for 21 days post installation. Thereafter, the animals in eachgroup were killed by an overdose of sodium pentobarbital (100-125 mg/kgip) and their lungs processed for bronchoalveolar lavage, biochemicaland histopathological studies.

Monoclonal Antibodies

Ha31/8 (hamster anti-α1β1 integrin IgG); Hal/29 (hamster anti-rat α2β1integrin IgG); and Ha4/8 (hamster anti-rat control IgG) are described inMendrick and Kelly, Lab. Invest. 69:690-702 (1993); Mendrick et al, Lab.Invest. 72:367-375 (1995), and commercially available (Pharmingen, SanDiego, Calif.).

Determination of Total Cell Number and Protein Levels in BroncoalveolarLavage

After cannulation of the trachea the lungs were lavaged with 5 ml ofisotonic saline, given in five aliquots of 1 ml. The saline wasadministered with a syringe through the cannula, the chest wall wasgently massaged, and the fluid was withdrawn. The fluid was centrifugedat 1500 g for 20 minutes at 4 degree C., and resusended in isotonicsaline solultion. The protein content for the supernatant frombroncoalveolar lavage specimens was detemined by a method of Lowry etal., J Biol. Chem. 1193: 265-275 (1951), with bovine serume albumin as astandard. Total leukocyte count of cells in suspension was determined ina Coulter Counter (Coulter Electronics, Hialeah, Fla.).

Determination of Hydoxyproline

The lungs of animals used for biochemical studies were perfused in situvia the right ventricle with ice-cold isotonic saline to wash out bloodfrom the pulmonary vasculature through an opening in the left auricle.The lung lobes were quickly dissected free of non-parenchymal tissue,dropped in liquid nitrogen for quick freezing and then stored at −80C.The frozen lungs were later thawed and homogenized in 0.1 M KCl, 0.02 MTris buffer (pH 7.6) with a Polytron homogenizer. Hydroxyproline contentof the lung homogenate as a measure of collagen content was quantitatedby the techniques of Woessner, Arch. Biochem. Biophys. 93: 440-447(1961).

Results

In this study, we tested the hypothesis that neutralizing antibody forintegrin α1β1 (antiα1β1) or integrin α2β1 (antiα2β1) may reducebleomycin (BL)-induced lung fibrosis in vivo. Male C57/BL6 mice wereintratraceally (IT) injected saline (SA) or BL at 0.08 U in 0.1 mlfollowed by intraperitoneal (IP) injection of the antibody (100 μg in0.2 ml) three times a week. Twenty-one days after the IT instillation,mice were killed for bronchoalveolar lavage (BAL), biochemical andhistopathological analysis.

Results: Hydroxy- Total BAL BAL Group Treatment proline cells protein(n) (IT + IP) (μg/lung) (×10⁻³/lung) (μg/lung) A(12) SA + PBS 231 ± 152.2 ± 0.14  171 ± 21 B(10) SA + IgG 225 ± 21 2.3 ± 0.24  167 ± 24 C(9)BL + IgG 371 ± 42* 6.7 ± 0.99* 1079 ± 292* D(11) BL + antiα1β1 230 ± 206.5 ± 1.17* 1238 ± 244* E(10) BL + antiα2β1 221 ± 14 4.2 ± 0.72  945 ±184**Significantly higher than other group

Lung histopathology showed fibrotic lesions in C group of mice, butlungs from D and E groups indicated somewhat reduced fibrosis comparedto C group. Our data demonstrated that treatment with antiα1β1 orantiα2β1 antibody reduced BL-induced lung collagen accumulation in mice.However, treatment with either antibody did not affect BL-inducedincreases in the BAL cell number and protein level, except for antiα2β1which reduced the total BAL cells. It is concluded that integrins α1β1and α1β1 play important roles in BL-induced pulmonary fibrosis and theuse of antiα1β1 or antiα2β1 antibody has great antifibrotic potential invivo.

Example 2

Histopathological Study

After lung lavage, the thoracic cavity was opened and the heart andlungs were removed en bloc. The lungs were instilled with a 1%glutaraldehyde-parafornaldehyde fixative in 0.12M cacodylate buffer at400 m Osm at 30 cm H₂O presure. The lungs are fixed via this pressurefor about 2 hours and then stored in fixative with the tracheasoccluded. Before embedding, the lung was isolated from the heart and allnon-pulmonary tissue by blunt dissection and removed. Blocks of tissuewere cut from at least two sagittal slabs (2-3 mm thick) from the rightcranial, right caudal, and left lung lobes of each lung. Each block wascut with about a 1 cm² face. The blocks were dehydrated in a gradedseries of ethanol and embedded in paraffin. Sections (5 μm thick) werecut from the paraffin blocks and stained with haematoxylin and eosin forhistological evaluations.

Data Analysis and Interpretation

The data are analyzed in terms of average values with their standarddeviations and standard errors of means. Student's t-test, chi-squaredistributions, correlation coefficient, analysis of variance (ANOVA) andmultiple comparison test will be applied to judge the significance ofdifferences between the control and treatment groups using a computerbased statistical package (SAS/STAT Guide, 6th Ed. Cary, N.C. pp.183-260 (1985)).

Histopathological Examination of Lungs

Histopathological examination of lungs was carried out on micesacrificed at 21 days after intratracheal instillation of saline orbleomycin. The mice treated with saline and control IgG (Group B) had novisible lesions and displayed interalveolar septa with a normal thinappearance. In contrast, mice treated with bleomycin and control IgG(Group C) had lesions varying from multifocal locations in proximalacini to a diffuse distribution that occasionally involved the pleura.In diffuse lesions, alveolar spaces were often obliterated by organizedconnective tissue and fibrotic lesions. In the multifocal lesioninteralveolar septa were thickened and lined by hypertrophied andhyperplastic cuboidal epithelial cells and abundant airway inflammatorycells. The lungs of mice treated with bleomycin and either anti-alpha1(Group D) and alpha2 (Group E) integrin antibodies appeared more likethose in Group B. Group D and E animals exhibited only a limited numberof fibrotic lesions, with mild multifocal septal thickening and smallaggregates of mononuclear cells.

Example 3

Cloning and mutagenesis of the α1-I domain. Human and rat α1β1 integrinI domain sequences were amplified from full length cDNAs (Kern, et al.(1994) J. Biol. Chem. 269, 22811-22816; Ignatius et al. (1990) J. CellBiol. 111, 709-720) by the polymerase chain reaction (PCR) (PCR COREKit; Boehringer Mannheim, GmbH Germany), using either human specific(5′-CAGGATCCGTCAGCCCCACATTTCAA-3′ [forward];5′-TCCTCGAGGGCTTGCAGGGCAAATAT-3′′ [reverse]) or rat specific(5′-CAGGATCCGTCAGTCCTACATTTCAA-3′ [forward];5′-TCCTCGAGCGCTTCCAAAGCGAATAT-3′ [reverse]) primers. The resulting PCRamplified products were purified, ligated into pGEX4t-i (Pharmacia), andtransformed into competent DH5α cells (Life Technologies). Ampicillinresistant colonies were screened for the expression of the ˜45 kDaglutathione S-transferase-I domain fusion protein. The sequences frominserts of plasmid DNA of clones that were selected for furthercharacterization were confirmed by DNA sequencing.

A rat/human chimeric α1-I domain (RΔH) was generated (MORPH Mutagenesiskit; 5 prime-3 prime), exchanging the rat residues G92, R93, Q94, andL97 (FIG. 4) for the corresponding human residues, V, Q, R, and R,respectively. Clones harboring the RΔH I domain were identified by theloss of a diagnostic Stu 1 restriction enzyme site, and the insertsconfirmed by DNA sequencing. The amino acid sequence of the human α1-Idomain is shown in FIG. 5.

Example 4

Generation of mAbs specific to the -α1 I domain. Monoclonal antibodieshave proved to be very useful probes in studying the relationshipbetween structure and function of integrin subunits. For example, mAbswere used extensively to study regions of the β1 subunit associated withan activated conformation (Qu, A., and Leahy, D. J. (1996) Structure 4,931-942). Thus, to identify potential probes for conformational changesof the α1-I domain, we generated a panel of mAbs to the human α1-Idomain.

Generation of anti-α1 I domain Monoclonal Antibodies. FemaleRobertsonian mice (Jackson Labs) were immunized intraperitoneally (i.p.)with 25 μg of purified human α1β1 (Edwards et al. (1995) J. Biol. Chem.270, 12635-12640) emulsified with complete Fruend's adjuvant (LifeTechnologies). They were boosted three times i.p. with 25 βg of α1β1emulsified with incomplete Freunds's adjuvant (Life Technologies). Themouse with the highest anti-α1-I domain titer was boosted i.p. with 100μg of α1β1 three days prior to fusion, and intravenously with 50 μg ofα1β1 one day prior to fusion. Spleen cells were fused with FL653 myelomacells at a 1:6 ratio and were plated at 100,000 and 33,000 per well into96 well tissue culture plates.

Supernatants were assessed for binding to the α1β1 integrin by singlecolor FACS. Prior to FACS analysis, supernatants were incubated withuntransfected K562 cells to eliminate IgG that bound solely to the βsubunit. Subsequently, 3-5 ×10 ⁴ K562 cells transfected with the α1integrin subunit (K562-α1) suspended in FACS buffer (1% fetal calf serum(FCS) in PBS containing 0.5% NaN₃) were incubated with supernatant for45 minutes at 40° C., washed and incubated with anti-mouse IgGconjugated to phycoerythrin. After washing twice with FACS buffer, cellswere analyzed in a Becton Dickinson Flow Cytometer.

Supernantants from the resulting hybridomas were screened for binding tothe α1-I domain. Briefly, 50 μl of 30 μg/ml human α1-I domain-GST fusionin PBS was coated onto wells of a 96 -well plate (Nunc) overnight at 4°C. The plates were washed with PBS, blocked with 1% BSA in PBS and thehybridoma supernatant was incubated with the I domain at roomtemperature for 1 hour. After extensive washing with PBS containing0.03% Tween 20, alkaline phosphatase linked anti-mouse IgG (JacksonImmunoResearch) was added for an additional hour. After a final wash, 1mg/ml p-nitrophenylphosphate (pNPP) in 0.1 M glycine, 1 mM ZnCl₂, and 1mM MgCl₂ was added for 30 minutes at room temperature, and the plateswere read at O.D. 405.

Selected supernatants were tested for their ability to inhibit K562-α1dependent adhesion to Collagen IV. K562-α1 cells were labeled with 2 mM2′,7′ (bis-2-carboxyethyl-5 and 6) carboxyfluorescein pentaacetoxymethylester (BCECF; Molecular Probes) in DMEM containing 0.25%BSA at 37° C. for 30 minutes. Labeled cells were washed with bindingbuffer (10 mM Hepes, pH 7.4; 0.9% NaCl; and 2% glucose) and resuspendedin binding buffer plus 5 mM MgCl₂ at a final concentration of 1×10⁶cells/ml. 50 μl of supernatant was incubated with an equal volume of2×10⁵ K562-α1 cells in wells of a 96 well plate. The plate was thencentrifuged and the supernatants removed. Cells were resuspended inbinding buffer and transferred to wells of a collagen-coated plate andincubated for 1 hour at 37° C. Following incubation, the non-adherentcells were removed by washing three times with binding buffer. Attachedcells were analyzed on a Cytofluor (Millipore).

We initially identified 19 hybridomas, the supernatants of which boundto human leukemia K562 cells expressing the alp integrin (K562-α1) andto the α1-I domain. The immunoglobulins were purified from each of thesehybridomas and tested for the ability to block either K562-α1 or α1-Idomain binding to collagen IV. The mAbs fall into two classes: thosethat block and those that do not block α1β1 function. For example, whilethe mAbs produced by clones AEF3, BGC5 and AJH10 bind the α1-I domain(FIG. 2A, data not shown for BGC5), only mAb AJH10 inhibits α1-Idomain-dependent (FIG. 2B) or K562-α1 (FIG. 2C) adhesion to collagen IV.

Sequencing of the Complementarity Determining Regions. To establish theclonal origin of this panel of mAbs, we amplified by PCR and sequencedthe CDRs from 12 of the 19 antibodies (data not shown).

2 μg of mRNA, isolated from 10⁷ hybridomas ( FastTrack mRNA isolationkit, Invitrogen), was reverse transcribed (Ready-To-Go You Prime FirstStrand Kit, Pharmacia Biotech) using 25 pM each of the followingprimers: heavy chain VH1FOR-2 (Michishita et al. (1993) Cell 72,857-867); light chain, VK4FOR, which defines four separate oligos (Kernet al. (1994) J. Biol. Chem. 269, 22811-22816). For each hybridoma,heavy and light chains were amplified in four separate PCR reactionsusing various combination of the following oligos: 1) Heavy chain:VH1FR1K (Kamata et al. (1995) J. of Biol. Chem. 270, 12531-12535), VH1BACK, VH1BACK (Baldwin et al.(1998) Structure 6, 923-935), V_(H)fr1a,V_(H)fr1b, V_(H)fr1e, V_(H)fr1f, V_(H)fr1g (Ignatius et al. (1990) J.Cell Biol. 111, 709-720), or VH1FOR-2 (Michishita, M., Videm, V., andArnaout, M. A. (1993) Cell 72, 857-867); 2) Light chain: VK1BACK(Baldwin et al. (1998) Structure 6, 923-935), VK4FOR, VK2BACK oligos(Kem et al. (1994) J. Biol. Chem. 269, 22811-22816), or V_(K)fr1a,V_(H)fr1c, V_(H)fr1e, V_(H)fr1f (Ignatius et al. (1990) J. Cell Biol.111, 709-720). Products were amplified (5 min at 95° C., 50 cycles of 1min at 94° C., 2 min at 55° C., 2 min at 72° C., and a final cycle of 10min at 72° C.), gel purified (QIAquick, Qiagen), and sequenced directlyusing various of the listed oligos on an ABI 377 Sequencer.

Sequences from clones producing function-blocking mAbs were nearlyidentical across all the complementarity-determining regions (CDRs) andthe intervening framework regions suggesting that these hybridomas areclonally related.

Example 5

Immunoblotting and FACS Analysis. Sequences of the variable regions ofthe non-blocking antibodies were markedly different from the clonallyrelated family of sequences found for the blocking antibodies. As theblocking antibodies appear to originate from a single clone, we choseone (AJH 10) to characterize further.

Immunoblotting The smooth muscle cell layer dissected from sheep aorta,and K562-α1 cells were extracted with 1% Triton X-100 in 50 mM Hepes, pH7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl flouride (PMSF), 20 μg/mlaprotinin, 10 μg/ml leupeptin, 10 mM ethylenediaminetetraacefic acid(EDTA). Samples were subjected to 4-20% gradient SDS-PAGE, andelectroblotted onto nitrocellulose membranes. The blots were blockedwith 5% dry milk in TBS; washed in TBS containing 0.03% Tween-20, andincubated with antibodies in blocking buffer containing 0.05% NaN₃ for 2hours. Blots were then washed as before, incubated with horseradishperoxidase conjugated anti-mouse IgG for one hour, washed again and thentreated with ECL reagent (Amersham). Blots were then exposed to film(Kodak) for 30 to 60 seconds, and developed.

Immunoblotting (FIG. 3A) and FACS analysis (FIG. 3B) demonstrate thatAJH10 reacts with human, rabbit, and sheep, but not rat α1β1 integrinsuggesting that the blocking mAbs bind to an evolutionarily conserved,linear epitope. The non-blocking mAbs were neither efficient atimmunoblotting nor did they react with species other than human.

Example 6

Binding of the α1-I domain to collagen is divalent cation-dependent

A. Purification of the α1-I domains.

The α1-I domains were expressed in E. coli as GST(glutathione-S-transferase) fusion proteins containing a thrombincleavage site at the junction of the sequences. The clarifiedsupernatant from cells lysed in PBS was loaded onto a glutathioneSepharose 4B column (Pharmacia) which was washed extensively with PBS.The α1-I domain-GST fusion protein was eluted with 50 mM Tris-HCl, pH8.0, 5 mM glutathione (reduced). For denaturation studies, the I domainwas cleaved with thrombin in 50 mM Tris, pH 7.5, and purified from theGST fusion partner. DTT was added to 2 mM and the sample was loaded on aglutathione Sepharose 4B column. The flow-through and wash fractionswere pooled and loaded onto a Q Sepharose FF column (Pharmacia). Theα1-I domain was eluted with 50 mM Tris HCl, pH 7.5, 10 mM2-mercaptoethanol, 75 mM NaCl. The purified I domain displayed itspredicted mass (Lee et al. (1995) Structure 3, 1333-1340, 871 Da) byelectrospray ionization-mass spectrometry (ESI-MS), migrated as a singleband by SDS-PAGE, and the protein eluted as a single peak of appropriatesize by size exclusion chromotography on a Superose 6 FPLC column(Pharmacia).

B. Functional Analysis

96 well plates were coated overnight at 4° C. with 1 μg/ml collagen IV(Sigma) or collagen Type I (Collaborative Biomedical), washed withTriton buffer (0.1% Triton X-100; 1 mM MnCl₂; 25 mM Tris-HCl; 150 mMNaCl), and blocked with 3% bovine serum albumin (BSA) in 25 mM Tris-HCl;150 mM NaCl (TBS). Serial dilutions of the α1-I domain-GST fusionprotein in TBS containing 1 mM MnCl₂ and 3% BSA were incubated on thecoated plates at room temperature for 1 hour, and washed in Tritonbuffer. Bound α1-I domain was detected with serial additions of 10 μg/mlbiotinylated anti-GST polyclonal antibody (Pharmacia);ExtrAvidin-horseradish peroxidase (Sigma) diluted 1:3000 in TBScontaining 1 mM MnCl₂ and 3% BSA, and 1-Step ABTS(2,2′-Azine-di[3-ethylbenzthiazoline sulfonate]; Pierce). Plates wereread at O.D. 405 on a microplate reader (Molecular Devices).

Results.

The human and rat (95% identity to human) α1-I domains were expressed inE. coli as GST-fusion proteins and purified over glutathione sepharose.Both proteins were examined for binding to collagen I and IV using avariation of an ELISA-based assay previously described (Qu, A., andLeahy, D. J. (1995) Proc. Natl. Acad. Sci USA 92, 10277-10281). Thehuman α1-I domain binds collagen IV with better efficiency than collagenI (FIG. 1A). An antibody specific to theα1-I domain, but not an antibodyspecific to the α2-I domain (FIG. 1B) abrogated binding to both ligands(data for collagen I is not shown). Both Mn²⁺ and Mg²⁺ stimulatedbinding, and EDTA reduced binding to background levels (FIG. 1C). Nomeasurable differences in ligand binding were detected between the humanand rat α1-I domains suggesting that the sequence differences betweenspecies are not functionally relevant (data not shown). Thus, the α1-Idomain, specifically, require cation for efficient ligand binding.

Example 7

A Cation-Dependent Epitope Resides near the MIDAS motif. We exploitedthe observation that AJH10 recognizes the human, but not the rat α1-Idomain sequences to map the epitope for the α1β1 function-blocking mAbs.The human and rat sequences differ by only 12 amino acids, 4 of whichlie in a stretch of 6 amino acids (aa 92-97, FIG. 4A) adjacent to thecritical threonine (FIG. 4A, aa 98) within the MIDAS motif To test thehypothesis that the 6 amino acid residues, Val-Gln-Arg-Gly-Gly-Arg,comprise the epitope for the blocking mAbs, we constructed a chimeric Idomain (RΔH), exchanging the rat residues G92, R93, Q94, and L97 for thecorresponding human residues, V, Q, R, and R, respectively. AJH 10,along with all the function-blocking mAbs, recognizes the chimeric Idomain (RΔH; FIG. 4B).

To orient these residues with respect to the MIDAS domain in thetertiary structure of the α1-I domain, we modeled the α1-I domain usingthe coordinates of the crystal structure of the α2 I domain.

A homology model of the human α1 I-domain was built using the X-raycrystal structure of the human α2 I-domain (Ward et al. (1989) Nature341, 544-546). The model was built using the homology modeling module ofInsight II (version 2.3.5; Biosym Technologies). The program CHARMM(Clackson et al. (1991) Nature 352, 624-628) was used with theall-hydrogen parameter set 22 with a distant dependent dielectricconstant of two times the atom separation distance. We first did 1000steps of steepest descent minimization with mass-weighted harmonicpositional constraints of 1 kcal/(mol Å²) on all atoms of the α1-Idomain. This minimization was followed by another 1000 steps of steepestdescent and 5000 steps of Adopted-Basis Newton Raphson with constraintsof 0.1 kcal/( mol Å²) on the C-α atoms of the α1-I domain to avoidsignificant deviations from the α2-I domain X-ray crystal structure.

The α1β1 and α2β1 integrin sequences exhibit 51% identity with noinsertions or deletions, suggesting that the overall structure of thetwo I domains will be similar. The metal coordination site is predictedto be the same in the α1-I domain as in the α2-I domain, and theresidues that comprise the epitope for the blocking mAbs lie on a loopbetween helix α3 and helix α4 which contains the threonine within theMIDAS motif critical for cation binding. The α1-I domain model predictsthat the amide nitrogen of Q92 (FIG. 4A) hydrogen bonds with thecarbonyl group of I133, the residue adjacent to S32. Thus, the loop thatcontains the epitope may play a functional role in stabilizing the MIDASregion.

The proximity to and the potential interaction of the loop containingthe epitope with the MIDAS motif suggested that the epitope, itself,might be sensitive to the presence of divalent cation. InitialELISA-based experiments confirmed that binding of AJH10, but not AEF3(FIG. 6A) to purified α1β1 integrin increases in the presence ofcations. Binding of AJH10 to cell surface-expressed α1β1 is alsoenhanced by the addition of cation (FIG. 6B). To further analyze thisobservation, we measured the relative binding affinities of the blockingmAbs, in the presence or absence of divalent cations, using a surfaceplasmon resonance (SPR) biosensor. Monitoring the reversible binding ofthe mAbs to the recombinant α1-I domain in real time allows thederivation of the association (ka) and dissociation rates (kd), as wellas the corresponding apparent dissociation constants (K_(D)) Theaddition of cation decreased the K_(D) of blocking mAb AJH10 from 400 to20 nM (data not shown). The addition of cation had no effect on theK_(D) of non-blocking, control mAb AEF3 (data not shown). Analysis ofthe ka and kd associated with binding reveals that the increase inaffinity is primarily attributable to a decrease in the rate ofdissociation (data not shown). For example, in the absence of cation,AJH10 has a dissociation rate constant of 1.65×10⁻³/sec. Addition ofMn²⁺ decreases the dissociation rate constant by a factor of 8 to2.12×10⁻⁴/sec (data not shown) while increasing the association rate byonly a factor of 2 (3.9×10³ M⁻s⁻¹ to 8.0×10³ M⁻¹ s⁻¹). Thus, theaddition of divalent cation appears to stabilize the epitope rather thanunmask a cryptic site, consistent with the proximity of the epitope tothe MIDAS region.

Example 8

Cation is required for I domain Stability. One interpretation of theeffect Mn²⁺ and Mg²⁺ have on epitope expression is that divalent cationsare required to stabilize the MIDAS region, or the entire α1-I domain.Thus, we looked at the stability of the α1-I domain in the presence orabsence of cations under denaturing conditions.

The presence of divalent cations had a stabilizing effect on the α1-Idomain structure readily detected by measuring the susceptibility of theprotein to denaturation by urea (FIG. 7).

The denaturation of the α1-I domain as a function of urea was measuredby fluorescence spectroscopy in an Aminco-Bowman series 2 LuminescenceSpectrometer. Samples containing 0.6 μM α1-I domain in 50 mM Tris HCl pH7.0, 0.15 mM DTT with no addition, 1 mM CaCl₂, or 1 mM MnCl₂ and withthe varying amounts of urea were analyzed at 25° C. using an excitationwavelength of 280 nm. Emission spectra from 300-400 nm were collected.Fluorescence data at 350 nm were plotted as a function of urea andstandardized using the change in fluorescence from 0 to 9 M urea foreach of the test conditions as a measure of the total fraction folded.

As described above, the denaturation of the α1-I domain was assessed bymonitoring the change in intrinsic fluorescence that results from theexposure of buried tryptophan and tyrosine residues to the aqueousenvironment as the protein unfolds. Denaturation produced both anincrease in fluorescence intensity and a red shift in the emissionspectrum. The maximal effect was seen at 360 nm where denaturation ofthe α1-I domain resulted in a greater than 4-fold increase in intrinsicfluorescence intensity. In the absence of divalent cation, the α1-Idomain was sensitive to the presence of low concentrations of urea andthe amount needed to produce a half maximal change in fluorescenceintensity was 3.4 M urea. In the presence of Mn²⁺, half maximaldenaturation shifted to 6.3 M urea, indicating a substantialstabilization of the α1-I domain.

The output of the spectrophotometric data discussed above is determinedprimarily by the fluorescence of a single buried tryptophan, which lieswithin the MIDAS region of the α1-I domain (W36, FIG. 4A). Thus, thespectrophotometric data only provide a view of the MIDAS region and notof the entire I-domain.

Example 9

Circular dichroism. To distinguish between local and possible wide rangeeffects on structure, we determined, by measuring circular dichroismspectra in the presence or absence of Mn²⁺ and Mg²⁺, the temperature atwhich the I domain denatured, and the effect of denaturation onsecondary structure of the protein. The circular dichroism measurementsdescribed below revealed that cation binding effects not just the localexpression of the epitope, but stabilizes the secondary structure of theα1-I domain.

Circular dichroism spectra were recorded using a J-710spectropolarimeter (JASCO, Japan) equipped with a programmabletemperature water bath (CTC-345, JASCO). Far-UV (185-250 nm) andtemperature-dependent measurements were performed using U-type cells ofpath-length 0.0148 cm and volume 0.045 ml with α1-I domain in 20 mMHEPES, 1mM EDTA, 1 mM DTT, pH 7.5 in the absence of divalent cations, orthe presence of either 2 mM Mg²⁺ or Mn²⁺ at a protein concentration of55 μM. CD spectra were recorded using a scan speed of 20 nm/min, aresponse time of 2 s and a band-width of 2 nm. Temperature-dependentmeasurements were performed in the range 10-80° C. The continuoustemperature scan at fixed wavelength (222 nm) in the far-UV range wasdone using a scan rate of 50° C./hr and a response time of 8 s. Data arepresented as molar ellipticity per residue.

Near and far UV CD spectra for the α1-I domain, in the presence andabsence of cation at room temperature, were indistinguishable (data notshown). In contrast, large, cation-dependent differences were seen inthe susceptibility of the I domain to thermal denaturation. In theabsence of divalent cations, the I domain denatured at T_(m)=49.5° C.Both Mn²⁺ (T_(m)=58.6° C.) and Mg²⁺ (T_(m)=54.6° C.) stabilized the Idomain as indicated by increases in T_(m) (FIG. 8). Heat denaturation inthe apo state was accompanied by a 20-25% decrease in ordered secondarystructure at 65° C. Decreases of 45% were observed for the Mn²⁺ state at70° C. and of 34% for the Mg²⁺ state at 80° C. For the apo state, CDspectrum at 65° C. and 80° C. have minima, which are characteristic of ahigh content of a helical structure whereas in the presence of divalentcations, CD spectra at 70-80° C. have shape that are characteristic for“aggregational” β-structure. These data suggest that, in addition to thelocal stabilizing effect cations have on the MIDAS region, the presenceof cations has a wide ranging effect on the secondary structure of theα1-I domain. It is interesting to note the Mn²⁺ is more stabilizing thanMg²⁺ as evidenced by a greated shift in T_(m). Since Mn²⁺ is moreeffective at promoting ligand binding to the α1β1 integrin (Bridges etal. (1995) Mol. Immunol 32, 1329-1338), the stabilizing effects of Mn²⁺may be related to the increased affinity of the I domain for ligand.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. A method for treating a subject suffering from a fibrotic condition,comprising administering to the subject a pharmaceutical composition,the pharmaceutical composition comprising an antibody molecule thatantagonizes an interaction of α1β1 with its ligand.
 2. A methodaccording to claim 1, wherein the antibody is selected from the groupconsisting a human antibody or a fragment of a human antibody, achimeric antibody or fragment of a chimeric antibody, or a humanizedantibody and fragments thereof or a fragment of a humanized antibody. 3.A method of claim 1, wherein the antibody is a monoclonal antibody.
 4. Amethod of claim 1, wherein the antibody or antibody fragment isadministered to the subject (a) around once or twice approximately everyseven days (b) in an amount of between about 0.3 mg/kg/day to about 5mg/kg/day.
 5. A method of claim 1, wherein the subject is a human. 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. A method of claim 1, wherein the composition isadministered parenterally.
 13. (canceled)
 14. (canceled)
 15. A methodfor treating a subject suffering from a fibrotic condition, comprisingadministering to the subject an antibody or fragment of an antibodyselected from the group consisting of AQC2 (ATCC PTA-3580); AJH10(ATCCPTA-3580); 1B3.1 (ATCC No. HB10336); or TS2/7.1 (ATCC No. HB-245).
 16. Amethod of claim 1, wherein the subject suffers from fibrosis of aninternal organ.
 17. A method of claim 16, wherein the internal organ isthe liver, kidney, heart blood vessels, or gastrointestinal tract.
 18. Amethod of claim 1, wherein the subject suffers from myelofibrosis, livercirrhosis, mesangial proliferative glomerulonephritis, crescenticglomerulonephritis, diabetic nephropathy, renal interstitial fibrosis,or HIV associated nephropathy.
 19. A method of claim 1, wherein thefibrosis is dermal fibrosis.
 20. A method of claim 19, wherein thesubject suffers from scleroderma, morphea, keloids, hypertrophic scars,familial cutaneous collagenoma, or connective tissue nevi of thecollagen type.
 21. A method of claim 1, wherein the fibrosis is fibrosisof the eye.
 22. A method claim 21, wherein the subject suffers fromdiabetic retinopathy, postsurgical scarring, or proliferativevitreoretinopathy